This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The main objective of this project is to establish the functional connection between folate and sphingolipid pathways and to explore underlying molecular mechanisms. Specifically, we propose to evaluate the role of sphingolipid signaling in mediation of the effects of folate deficiency and responses to the disturbance of intracellular folate metabolism. At present, there is a lack of knowledge regarding functional interactions between the folate and sphingolipid pathways, but there are specific indications that the two pathways are functionally connected. (1) They both rely on serine: as the principal one-carbon group donor into the folate pool and a substrate for de novo sphingolipid biosynthesis;(2) sphingolipid pathways depend on methylation, directly (methylation of phosphatidylethanolamine to phosphatidylcholine) and indirectly (regulation of enzyme expression via DNA/histones methylation), while folates are crucial for cellular methylation;(3) cross talk between the two pathways is suggested by experiments with fumonisine, an inhibitor of ceramide synthase, as well as in experiments with antifolates;(4) our studies have demonstrated significant changes in the levels of ceramides upon alterations of intracellular folate metabolism. Therefore, we have hypothesized that activation of sphingolipid pathways is the down-stream event of the folate stress. The alteration of methylation in sphingolipid pathways is proposed as the underlying mechanism in this process. Aims to test the hypotheses are: Aim 1. Determine effects of folate deficiency on sphingolipid pathways. Aim 2. Explore the role of folate-dependent methylation in sphingolipid pathways. Aim 3. Determine whether ceramide signaling is a component of cellular response to impaired folate metabolism.